It is now clear that antiretroviral therapy (ART) alone does not eradicate HIV: Even after more than 15 years of intensive and continuous therapy, the spread of the virus resumes within a few weeks upon cessation of ART in all but exceptional cases. A small pool of latently infected cells, usually referred as the reservoir of HIV infectio, provides a long-lived source of rebound viremia. Eradication of HIV has been hampered by the lack of information regarding the specific subsets harboring the viral reservoir and the mechanisms associated with the maintenance of latency. We have previously demonstrated that HIV persists in three memory T cell subsets displaying distinct functional and survival capacities, namely central (TCM) transitional (TTM) and effector (TEM) memory CD4+ T cells in the blood of virally suppressed subjects. Interestingly, our preliminary data indicate that these cell subsets also ensure HIV persistence in tissue reservoirs such as the lymph nodes, rectum, colon and terminal ileum. Therefore, there is an urgent need to better characterize these three cellular HIV reservoirs to achieve their eradication. The objective of this project is to identify mechanisms of action that can lead to the reactivation and subsequent elimination of latently infected cells in virally suppressed subjects. We hypothesize that the mechanisms responsible for HIV latency in TCM, TTM and TEM are different, and that these reservoirs should be targeted by different classes of anti-latency agents. We will first identify the molecular mechanisms responsible for HIV latency in TCM, TTM and TEM cells (Specific Aim 1). We will focus on epigenetic modification of the DNA through methylation and histone deacetylation, limited availability of critical transcription factors and inefficient elongation of the nascent vial transcripts. We will also use RNA sequencing to identify novel cellular pathways that correlate with the magnitude of the latent reservoir in the CD4+ T cell subsets from 24 virally suppressed subjects. We will then select the most efficient combination of compounds that will interfere with these mechanisms and allow elimination of all cellular reservoirs (Specific Aim 2). We have selected molecules that have shown promising effects in previous studies and/or that are currently tested in clinical trials. As it is highly likely that targeting a single cellular reservir or a single mechanism involved in viral latency will not be successful, we will test the ability of combinations of lead compounds from each class to disrupt latency in latently infected CD4+ T cells from virally suppressed subjects. Using a novel co-culture assay recently developed at VGTI Florida, we will assess if reactivation with this combination of anti-latency drugs can lead to the elimination of reservoir cells by autologous HIV specific CTL. The deliverable of this research project is a combination of 3-5 clinically tolerable agents that can reverse latency in authentic latently infected cells from virally suppressed subjects. The results from these studies will constitute the basis for the design of a clinical trial aimed at eradicating HIV.